Although large numbers of candidate biomarkers for malignant (Etzioni, et al., Nat. Rev. Cancer 3:243-52 (2003)), autoimmune (Prince, H. E., Biomarkers 10(Suppl. 1):44-9 (2005)), infectious (Zhang, et al., Science 298:995-1000 (2002)), neurological (Carrette, et al., Proteomics 3:1486-94 (2003)), metabolic (Roelofsen, et al., J. Cell Biochem. 93:732-40 (2004); Schwegler, et al., Hepatology 41:634-42 (2005)), and cardiovascular (Kittleson and Hare, Cardiovasc. Med. 15:130-8 (2005)) diseases have been generated using various high-throughput platforms (Omenn, et al., Proteomics 5:3226-45 (2005); Ping, et al., Proteomics 5:3506-19 (2005)), the production of high affinity antibodies necessary for biomarker validation remains slow and costly.
For example, CA125 is the most extensively studied biomarker for possible use in the early detection of ovarian carcinoma (Kenemans, P., et al., Eur. J. Obstet. Gynecol. Reprod. Biol. 49(1-2):115-24 (1993); Tamakoshi, K., et al., Gynecol. Obstet. Invest. 39(2):125-9 (1995); Dorum, A., et al., Eur. J. Cancer 32A(10):1645-51 (1996); Eagle, K., et al., Oncologist 2(5):324-329 (1997); Fures, R., et al., Coll. Anthropol. 23(1):189-94 (1999); and Jacobs, I. J., et al., Mol. Cell Proteomics 3(4):355-66 (2004)). CA125 is a mucin-like protein of high molecular mass, estimated from 200 to 20,000 kDa, although smaller subunits have been reported (O'Brien, T. J., et al., Int. J. Biol. Markers 13(4):188-195 (1998); Yin, B. W., et al., J. Biol Chem 276(29):27371-5 (2001)). CA125 cell surface expression is upregulated when cells undergo metaplastic differentiation into a Mullerian-type epithelium (Feeley, K. M., et al., Histopathology 38(2):87-95 (2001)). Although CA125 is conserved in mammals (Nouwen, E. J., et al., Differentiation 45(3):192-8 (1990); McDonnel, A. C., et al., Reproduction 126(5):615-20 (2003); Duraisamy, S., et al., Gene (2006)), there is no available antibody against mouse CA125 which impairs ovarian cancer studies in vivo.
New methods such as high-throughput screening by flow sorting of recombinant antibodies (scFv) displayed by yeast (Feldhaus et al., Nat. Biotechnol. 21:163-70 (2003)) can accelerate the identification of antigen specific scFv. However, yeast display scFv need to be converted into soluble and labeled recombinant antibodies for further use in immunoassays.
Biotinylation is a robust labeling method that modifies the lysine residues of target proteins and is routinely achieved in vitro with chemical agents (Bayer and Wilchek, Methods Enzymol. 184:138-60 (1990)). The extraordinarily high affinity binding of biotin to streptavidin (dissociation equilibrium constant or Kd=10−15M) (Green and Melamed, Biochem. J. 100:614-21 (1966)) and extremely slow dissociation rate have been used widely in biochemistry and molecular biology. However, chemical biotinylation is not specific and thus can inactivate lysine residues critical for protein function and conformation (Smith, et al., Nuc. Acids Res. 26:1414-20 (1998)). In contrast, in vivo biotinylation is a rare and highly specific post-translational modification (Cronan, J. E., J. Biol. Chem. 265:10327-33 (1990)) mediated by endogenous biotin ligases. Specific biotinylation of proteins and recombinant antibodies has been obtained in E. coli in vivo (Kipriyanov, et al., Protein Eng. 9:203-11 (1996); Sibler, et al., J. Immunol. Methods 224:129-40 (1999); Santala and Lamminmaki, J. Immunol. Methods 284:165-75 (2004); Warren, et al., Clin. Chem. 51:830-8 (2005)) and in vitro with purified biotin ligase (Saviranta, et al., Biocong. Chem. 9:725-35 (1998); Cloutier, et al., Mol. Immunol. 37:1067-77 (2000)). However, current in vivo biotinylation protocols can only label one protein at a time through lengthy molecular engineering that is not compatible with high-throughput approaches.